A quantitative method called isoTOP-ABPP (isotopic Tandem Orthogonal Proteolysis – Activity-Based Protein Profiling) was developed to profile the intrinsic reactivity of cysteine residues in native proteomes. This method uses an electrophilic iodoacetamide (IA) probe to label cysteine residues, which are then conjugated to a biotinylated TEV-protease recognition peptide for streptavidin enrichment. Isotopically labeled valine is used for quantitative mass spectrometry to measure IA-labeled peptides. The method enables the identification and quantification of cysteine reactivity across multiple proteomes. Hyperreactive cysteines, which show enhanced reactivity, were found to be associated with various functional roles, including catalytic activities, redox regulation, and sites of oxidative modification. These cysteines were identified in proteins of uncharacterized function, including a conserved residue in eukaryotic phylogeny that is essential for yeast viability and involved in iron-sulfur protein biogenesis. The method was also applied to computationally designed proteins, where it successfully distinguished catalytically active from inactive cysteine hydrolase designs. The study demonstrated that hyperreactive cysteines are intrinsic properties of residues and their local protein environments, not dependent on specific cell or tissue features. IsoTOP-ABPP ratios were independent of protein abundance or peptide ion intensity, indicating they are not influenced by MS-based ionization sources. The method was validated using human and mouse proteomes, revealing consistent isoTOP-ABPP ratios for individual cysteine residues. Hyperreactive cysteines were found to be functionally significant, often corresponding to active-site residues or sites of post-translational modification. For example, in the protein PRMT1, a hyperreactive cysteine was shown to be modified by oxidative stress and regulated by HNE. Similarly, other hyperreactive cysteines were identified as sites for glutathioneylation and nitrosylation. The study also showed that hyperreactivity can predict cysteine function in both native and designed proteins. In designed proteins, hyperreactive cysteines were associated with catalytic activity, suggesting that heightened nucleophilicity is a key feature of active catalysts. The isoTOP-ABPP method provides a versatile and high-throughput way to evaluate protein designs and has the potential to be used for screening novel cysteine-dependent enzymes. The method's ability to profile cysteine reactivity in complex biological systems, including living cells, and its ability to distinguish probe-accessible cysteines, makes it a valuable tool for studying protein function and regulation. The findings suggest that cysteine nucleophilicity may have been selected for during evolution to provide points of protein control by oxidative stress pathways. The study highlights the importance of hyperreactive cysteines in mammalian biology and their potential rolesA quantitative method called isoTOP-ABPP (isotopic Tandem Orthogonal Proteolysis – Activity-Based Protein Profiling) was developed to profile the intrinsic reactivity of cysteine residues in native proteomes. This method uses an electrophilic iodoacetamide (IA) probe to label cysteine residues, which are then conjugated to a biotinylated TEV-protease recognition peptide for streptavidin enrichment. Isotopically labeled valine is used for quantitative mass spectrometry to measure IA-labeled peptides. The method enables the identification and quantification of cysteine reactivity across multiple proteomes. Hyperreactive cysteines, which show enhanced reactivity, were found to be associated with various functional roles, including catalytic activities, redox regulation, and sites of oxidative modification. These cysteines were identified in proteins of uncharacterized function, including a conserved residue in eukaryotic phylogeny that is essential for yeast viability and involved in iron-sulfur protein biogenesis. The method was also applied to computationally designed proteins, where it successfully distinguished catalytically active from inactive cysteine hydrolase designs. The study demonstrated that hyperreactive cysteines are intrinsic properties of residues and their local protein environments, not dependent on specific cell or tissue features. IsoTOP-ABPP ratios were independent of protein abundance or peptide ion intensity, indicating they are not influenced by MS-based ionization sources. The method was validated using human and mouse proteomes, revealing consistent isoTOP-ABPP ratios for individual cysteine residues. Hyperreactive cysteines were found to be functionally significant, often corresponding to active-site residues or sites of post-translational modification. For example, in the protein PRMT1, a hyperreactive cysteine was shown to be modified by oxidative stress and regulated by HNE. Similarly, other hyperreactive cysteines were identified as sites for glutathioneylation and nitrosylation. The study also showed that hyperreactivity can predict cysteine function in both native and designed proteins. In designed proteins, hyperreactive cysteines were associated with catalytic activity, suggesting that heightened nucleophilicity is a key feature of active catalysts. The isoTOP-ABPP method provides a versatile and high-throughput way to evaluate protein designs and has the potential to be used for screening novel cysteine-dependent enzymes. The method's ability to profile cysteine reactivity in complex biological systems, including living cells, and its ability to distinguish probe-accessible cysteines, makes it a valuable tool for studying protein function and regulation. The findings suggest that cysteine nucleophilicity may have been selected for during evolution to provide points of protein control by oxidative stress pathways. The study highlights the importance of hyperreactive cysteines in mammalian biology and their potential roles